MC-based hydrogels were analyzed to investigate the rheological behavior and thus optimize the printing process parameters. Cells were tested in vitro on ring-shaped printed hydrogels; bulk MC hydrogels were used for comparison. In vitro tests used murine embryonic fibroblasts (NIH/3T3) and endothelial murine cells (MS1), and the resulting cell sheets were characterized analyzing cell viability and immunofluorescence. In terms of CS preparation, 3D printing proved to be an optimal approach to obtain ring-shaped CS. Cell orientation was observed for the ring-shaped CS and was confirmed by the degree of circularity of their nuclei: cell nuclei in ring-shaped CS were more elongated than those in sheets detached from bulk hydrogels. The 3D printing process appears adequate for the preparation of cell sheets of different shapes for the regeneration of complex tissues.A possible strategy in regenerative medicine is cell-sheet engineering (CSE), i.e., developing smart cell culture surfaces from which to obtain intact cell sheets (CS). The main goal of this study was to develop 3D printing via extrusion-based bioprinting of methylcellulose (MC)-based hydrogels. Hydrogels were prepared by mixing MC powder in saline solutions (Na Tissue engineering (TE) is one possible approach in regenerative medicine; its main goal is to overcome limited donor availability and the high risk of rejection, which affects the clinical treatments currently used ,2,3.3), can be mixed in aqueous solution to obtain thermo-responsive hydrogels [Among possible TE approaches, cell-sheet engineering (CSE) provides an approach that is innovative compared to the use of biodegradable scaffolds or the injection of cell suspensions at the body site, including by a cell carrier. CSE entails developing smart cell culture surfaces, permitting the in vitro culture of cells and the detachment by specific stimuli  of intact cell sheets (CS), preserving the gap junctions between cells and the extracellular matrix (ECM) proteins . These sydrogels ,8,9,10. In the CSE approach, MC-based hydrogels are exploited as substrates to produce CS in vitro. In particular, cells adhere and proliferate on the hydrogel’s hydrophobic surface (in its gel state) at 37 °C (T > LCST). On lowering the temperature (T < LCST), the hydrogel surface turns hydrophilic (in its sol state) and the cell sheets spontaneously detach . In addiThanks to recent developments in 3D printing technologies, hydrogel 3D printing is now under investigation with the goal of obtaining complex and reproducible shapes of the printed structures. With this approach, hydrogels or cell-laden hydrogels  can be printed, layer by layer, to produce engineered constructs ,16. The In this work, to the best of our knowledge, MC hydrogels were 3D printed for the first time in order to use them for the biofabrication of oriented CS. Accordingly, two MC-based hydrogels were prepared using the same MC concentration and different saline solutions and were applied with the aim of obtaining 3D-printed CSE substrates via extrusion-based bioprinting. After physical characterization, the resulting 3D-printed substrates were tested with N1H/3T3 murine fibroblasts and MS1 murine endothelial cells for the production of CS having ring-shaped geometry.w/v aqueous solution at 20 °C) was kindly supplied by The Dow Chemical Company . All basic chemicals were from Sigma-Aldrich  unless stated otherwise.Methylcellulose  or in 20 g/L phosphate buffered saline (PBS) (MC-PBS20). MC powder was added after heating the saline solutions to 55 °C to ensure a homogeneous powder dispersion. The resulting polymeric suspensions , using flat-plate geometry . A homemade isolation chamber in polymethyl methacrylate  was designed and fixed to the rheometer to prevent dehydration of the hydrogel under test.For each MC hydrogel composition, tests were run using three specimens taken immediately after 24 h hydration at 4 °C. The linear viscoelastic region (LVR) was identified at 20 and at 37 °C by applying a strain sweep between 0.1 and 100%, frequency 1 Hz. To determine the LCST, temperature sweep tests were then performed between 10 and 60 °C, applying a temperature ramp of 2 °C/min, 0.5% strain , frequency 1 Hz. The LCST was identified for each hydrogel composition, as reported elsewhere , from st®, Wacker, Munich, Germany) supports  of 1.45 μm/s, was chosen to achieve the deposition of a continuous strand of hydrogel.MC-based hydrogels were 3D-printed by an extrusion-based 3D printer  using a modified version of the printer filament specifically adapted to print hydrogels . Briefly, the Kiwi 4D printer allows the lodging of a 60 mL medical syringe into a cylindrical heating jacket, used to control the printing temperature depending on hydrogel characteristics. The extrusion process is controlled by a stepping motor connected to a piston that drives the plunger of the syringe. MC-specimens were 3D-printed directly on polydimethylsiloxane  of MC-based hydrogels were obtained by printing one layer 1 mm in height.n = 3) were weighed at the start of the test and incubated in 4 mL distilled water. At different time points , excess water was removed, the weights of the specimens were recorded, and 4 mL of fresh distilled water was added. The weight variation (ΔW) was calculated as follows:t and The stability of the bulk (MC-PBS20-bulk and MC-Na005-bulk) and printed (MC-PBS20-print and MC-Na005-print) MC-based hydrogels was evaluated in distilled water at 37 °C for up to 7 days. The hydrogel specimens (n = 3) were prepared by adding MC solutions in a tissue culture multiwell plate . The print-ring specimens were printed with the Kiwi 4D printer under a laminar flow cabinet; the printer was moved under the cabinet and disinfected with 70% v/v ethanol solution and sterilized by UV light for 30 min before MC printing. In the in vitro tests, murine embryonic fibroblasts  modified with a gene for the expression of green fluorescent protein (GFP) and endothelial murine cells  were selected to investigate the feasibility of using different cell phenotypes to produce CS. For both cell lines, Dulbecco Modified Eagle’s Medium  supplemented with fetal bovine serum , L-glutamine (1% v/v), Hepes (1% v/v), sodium pyruvate , and a solution of penicillin/streptomycin (1% v/v) was used.For the in vitro biological tests, two differently shaped specimens were tested: Bulk and print-ring specimens. MC-based hydrogels were prepared using a filter-sterilized solution  and UV light sterilized MC powder. Bulk specimens  assay. For all possible combinations of MC-based hydrogel (5 cells/well) onto each specimen and the culture plates were stored in an incubator  for 24 or 48 h. Tests were performed in triplicate. Cells cultured on TCPS alone at the same density were used as control.Previous in vitro cytotoxicity tests  had founhydrogel /specimen2), the plates were removed and placed at 4 °C for 20 min. Once spontaneously detached from the hydrogel surface, cell sheets were sucked up with a sterile pipette and moved onto a fresh TCPS plate for subsequent characterization by immunofluorescence analysis. Tests were performed in triplicate. For all possible combinations of MC-based hydrogel/specimen shape/cell phenotype, a cell sheet specimen was used for immunofluorescence analysis. All detached cell sheets were transferred onto electrostatic slides and stained with phalloidin to stain the actin filaments and with 4′,6-diamidino-2-phenylindole (DAPI) to co-stain the nuclei. The specimens were then observed through a fluorescence microscope . Images were then processed  to quantify the mean circularity of the nuclei in each type of cell sheet. Circularity is expressed by mean of a numerical value between 0 and 1, where 1 meant a perfect circle and 0 a stretched polygon, as described by Altomare et al. [Following the procedure described above, MC hydrogel samples (bulk and printed specimens) were prepared for cell sheet detachment tests. After 48 h of culture in the incubator  in order to qualitatively evaluate cell sheet adhesion and, hence, cell proliferation on the new substrate. As described elsewhere , a few d+ (fibroblasts) cells were seeded onto 3D-printed supports filled with both MC-Na005 and MC-PBS20 hydrogels. Cells were seeded in a 50:50 ratio (1.5 × 105 cells/support) and cultivated for 48 h. Co-cultured cell sheets were then detached by lowering the temperature as described above  and NIH-GFPed above , collect+ fibroblasts and (ii) MS1 endothelial cells were produced separately onto 3D-printed molds and filled with either MC-Na005 or MC-PBS20 hydrogels. NIH-GFP and MS1 CS were then detached and superimposed to obtain a sandwich comprising three layers in the order NIH–MS1–NIH. After 48 h of direct layer-to-layer contact, three-layered cell sheets were stained with α-SMA, GFP, and DAPI to detect the presence and the interaction of the three layers.To test the possibility of biofabricating complex multi-layered proto-tissues in vitro, CS made of (i) NIH-GFPExperiments were performed using at least three replicates for each assay. Data have been expressed as the mean ± standard deviation and compared statistically by means of the two-sample t-test , evaluating the normal distribution by the Shapiro–Wilke test.2SO4 and PBS concentrations were selected to achieve a salting-out effect, thus reducing the LCST of MC aqueous solutions to 37 °C, suitable for applications in the field of CSE [4− ions (deriving from Na2SO4 dissociation) and Cl− ions (deriving from KCl and NaCl dissociation), respectively, inducing water molecule spillage from the polymeric structure [3) are exposed and associate hydrophobically to form a physically cross-linked structure [Based on previous research ,22, the d of CSE ,22. LCSTtructure ,22,24. Mtructure . In parttructure ,9,10. Thtructure ,25,26. HThe LVR region of MC-based hydrogels was identified by means of strain sweep tests. The curves at 20 °C a of both3 groups of the MC thus leads to the association of the hydrophobic polymer chains of MC [plateau. The LCST values were obtained from the G’/T and η*/T curves . To the authors’ knowledge, this is the first time that MC-based hydrogels have been printed. In other studies reported in the literature, MC has been blended with other natural polymers for the printing process, rather than for the production of CS. Schutz et al. [After parameter optimization, due to the novelty of the technique, MC hydrogel rings were successfully printed with both MC-based formulations within the PDMS supports. A representative image of the hydrogel printed ring is shown in z et al.  reportedz et al.  or to imz et al. .p < 0.05) in weight variation during swelling (days 1–4), following a similar trend for the two types of specimens. In the case of the MC-PBS20 hydrogel (p < 0.05). The larger weight variation of the MC-based printed hydrogels may have been caused by the shear stress induced by the printer needle on the gel during the printing process. As a consequence of extrusion, the MC chains in the printed hydrogel align and approach so closely to one another that the effect is comparable to an increase in concentration of MC in the gel: An increase in MC concentration determines an increased swelling of MC-based hydrogels, as reported by Thirumala et al. [The weight variation trend over time (up to 7 days) was similar for the two MC hydrogel formulations analyzed, both before and after printing . In parthydrogel b, there a et al. .p < 0.05) for MC-Na005 bulk and MC-PBS20 bulk and printed ring specimens. Thus, the 3D printing technique did not affect cell viability of the resulting CS. Comparing the two hydrogels (MC-Na005 versus MC-PBS20), no significant differences emerged between the two formulations either for bulk or for printed ring specimens.The viability of murine fibroblasts and murine endothelial cells was evaluated after 24 and 48 h in culture on the MC-based hydrogels under study. For bulk and printed ring specimens, the alamarBlue assay showed increased fluorescence values  between For all combinations of MC-based hydrogel formulation/specimen type/cell phenotype, CS were detached after 48 h of culture. Complete and ready detachment of CS from the surface of the hydrogels was achieved by placing the culture plates at 4 °C for 20 min. With a decrease in temperature, as was said above, the structure of the MC-based hydrogels changed to a hydrophilic sol state. Thus, the cells spontaneously detached from the hydrogel without the need for proteolytic enzymes . For each type of sample, the efficiency of the detachment operation has been expressed as the number of intact detached cell sheets versus the total number of seeded hydrogels a. All CSFluorescence images acquired for GFP , phalloidin (red), and DAPI (blue) staining highlight the cell organization on the bulk and the printed ring MC-based hydrogels. CS produced on bulk hydrogels c were cop < 0.05), except in the case of the MC-PBS20 hydrogels cultured with MS1 endothelial cells , aimed at quantifying the circularity of cell nuclei present on each cell sheet, showed that the mean circularity values for bulk sheets were close to 1 (indicating perfect circularity) whereas those for the printed ring differed significantly  multi-species and (ii) multi-layer CS. The results are shown in +) were seeded together, mixing them in a 50:50 ratio to avoid any predominant population (5/support) on a collagen coating and detached after 48 h by cooling the system, affording hydrogel phase transition. As shown in To obtain multi-species CS, endothelial cells (MS1) and fibroblasts (NIH-GFPpulation a,b. To o+ CS were produced separately on 3D-printed supports, detached by cooling the hydrogel, and collected. Successively, using MS1 and NIH CS, a three-layer sandwich was produced by superimposing CS made of NIH–MS1–NIH cells, respectively (The possibility of interactions occurring between detached CS of different origins was also investigated. MS1 and NIH-GFPectively d,e. The Two MC-based hydrogels were characterized for their possible application in the field of CS engineering. The possibility of printing MC-based hydrogels by an extrusion-based printing technology was investigated for the first time and successfully applied. The resulting CS showed that the 3D printing technique is a promising strategy to obtain CS that replicate the geometry of the printed MC hydrogel substrate. This allows the acquisition of more complex-shaped CS, compared to those obtained by culturing cells on MC hydrogels poured into simple-shaped molds. The cell orientation in the ring-shaped CS, confirmed by the degree of circularity of the nuclei, was of particular interest.


Actinopyga lecanora) protein using bromelain and stabilized by their encapsulation in chitosan (chit) nanoparticles (NPs). The nanoparticles were characterized for in vitro physicochemical properties and their antihypertensive effect was then evaluated on spontaneously hypertensive rats (SHRs). The results of a physicochemical characterization showed a small particle size of 162.70 nm, a polydispersity index (pdi) value of 0.28, a zeta potential of 48.78 mV, a high encapsulation efficiency of 75.36%, a high melting temperature of 146.78 °C and an in vitro sustained release of the biopeptides. The results of the in vivo efficacy indicated a dose-dependent blood pressure lowering effect of the biopeptide-loaded nanoparticles that was significantly higher (p < 0.05) compared with the un-encapsulated biopeptides. Moreover, the results of a morphological examination using transmission electron microscopy (TEM) demonstrated the nanoparticles as homogenous and spherical. Thus, the ACE-inhibitory biopeptides stabilized by chitosan nanoparticles can effectively reduce blood pressure for an extended period of time in hypertensive individuals.Recent biotechnological advances in the food industry have led to the enzymatic production of angiotensin I-converting enzyme (ACE)-inhibitory biopeptides with a strong blood pressure lowering effect from different food proteins. However, the safe oral administration of biopeptides is impeded by their enzymatic degradation due to gastrointestinal digestion. Consequently, nanoparticle (NP)-based delivery systems are used to overcome these gastrointestinal barriers to maintain the improved bioavailability and efficacy of the encapsulated biopeptides. In the present study, the ACE-inhibitory biopeptides were generated from stone fish ( Hypertension is defined as a sustained increase in systolic blood pressure greater than 140 mmHg and/or diastolic blood pressure less than 90 mmHg. It is inferred as a common public health risk of global concern that is associated with cardiovascular-related diseases such as heart failure, kidney failure, stroke and disability .Cardiovascular diseases (CVDs) have been identified as the major cause of mortality, with a global death rate of greater than 17.3 million cases in 2013, which accounted for 31% of all the reported deaths during the year . The ratMore importantly, the suppression of hypertension/elevated blood pressure alone among other risk factors could sufficiently reduce mortality due to CVDs by 30.4% and 38.0% in males and females, respectively .Presently, ACE-inhibitors, renin inhibitors, aldosterone inhibitors, beta blockers and angiotensin receptor blockers are the five major classes of antihypertensive drugs with specific target site inhibitory action on the renin–angiotensin–aldosterone system (RAAS) that are used in the management of hypertension. However, the therapeutic potential of such drugs is limited by their cost and related adverse effects, including insulin resistance and diabetes, skin rashes and taste disturbance, etc. ,9.Alternatively, ACE-inhibitory biopeptides generated through the enzymatic hydrolysis of food proteins have been reported to exhibit blood-pressure-lowering capacities in hypertensive animals with little or no side effects ,11,12,13Several approaches including permeation enhancers, enzyme inhibitors and different polymeric carrier systems have been used to overcome these challenges ,16. In tBy virtue of their large surface-area-to-volume ratio, small-sized nanocapsules have been reported to circumvent first pass effects and to improve the solubility, gastrointestinal stability, target site delivery, bioavailability and biological role of the encapsulated bioactive agent . In due Chitosan has been safely used as a pH sensitive, biodegradable, biocompatible and mucoadhesive polymer in the fabrication of polymeric nanoparticles for oral-colon delivery and the controlled release of bioactive peptides and proteins ,21,22.Chitosan-based nanoparticles have been reported to enhance the oral delivery and bioavailability of anti-cancer agents ,24,25,26In the present study, chitosan nanoparticles were produced and evaluated as potential oral delivery vehicles for the ACE-inhibitory biopeptides derived from stone fish protein using bromelain. The biopeptides were nanoencapsulated using chitosan by the ionotropic gelation method and characterized in vitro for physicochemical properties. The antihypertensive efficacy of the chitosan nanoparticles containing biopeptides was then studied on spontaneously hypertensive rats.The Chitosan-AntBiop-NPs were fabricated by a simple technique  under opAs shown in In The optimized condition to fabricate the Chitosan-AntBiop-NPs was established to include a AntBiop:Chitosan mass ratio of 0.35, stirring speed of 8000 rpm and a stirring time of 25 min. The Chitosan-AntBiop-NPs prepared under these conditions showed a high EE of 75.36%. The NPs also have a small particle size of 162.70 nm, a low pdi value of 0.28, and a high positive zeta potential  of 48.78 mV .The increased chitosan concentration caused a decreased shear effect, which lead to a highly viscous formulation characterized by capsules of large particle size and high EE. However, the tween 80 that was used as an emulsifier to sterically stabilize the nanocapsules induced reduced surface tension and improve the shear effect, resulting in smaller-sized nanocapsules while still maintaining a high EE.As previously reported, the size of the chitosan nanoparticles increased with the increase in concentration of chitosan , and a hThe stirring speed and stirring time could affect particle size and the rate at which chitosan formed a viscous gel to ionically gelate the AntBiop. Thus, the increase in these factors and the amount of cross-linking agent, sodium tripolyphosphate, were found to maximize the incorporation and the entrapment efficiency of the AntBiop in the Chit-AntBiop-NPs and to lead to smaller sized nanocapsules. In a similar finding, the stirring speed and stirring time were optimized to produce chitosan nanoparticles with a small particle size and high EE . HoweverIn contrast, we were able to fabricate nanocapsules of much smaller particle size with high entrapment efficiency and less stirring time.As shown in The in vitro release profile of Chitosan-AntBiop-NPs is shown in The in vitro release showed a prolonged and sustained release of the AntBiop from the Chit-AntBiop-NPs into the suspending medium. This indicated the potential oral application of the Chit-AntBiop-NPs for preventing gastrointestinal degradation and improving sustained release and prolonging the therapeutic effect of the AntBiop as an antihypertensive agent.The thermal behavior of the blank chitosan and Chitosan-AntBiop-NPs are presented in Similarly, the high melting temperature was reported to be due to the interaction between the active agent and its coating polymer, such that more energy is needed to dissipate their cohesive forces than that required to break the blank capsule .As shown in p < 0.05) reduced systolic blood pressure (SBP) by 19.78 ± 9.966 mmHg at 2 h and 11.78 ± 8.483 mmHg at 4 h respectively (Group II).As shown in p < 0.05) in SBP between 2 and 8 h post oral administration was observed for 50 mg/kg captopril and for the 200, 400 and 800 mg/kg Chit-AntBiop-NPs. The maximum SBP-lowering effects of captopril and the Chit-AntBiop-NPs were 50.18 ± 8.90 mmHg and 59.78 ± 7.68 mmHg at 6 h post administration. Thereafter, the SBP increased gradually towards the untreated levels. A sustained decrease in SBP was still observed for the given doses of unencapsulated biopeptides, captopril and different doses of Chit-AntBiop-NPs even at 24 h of post administration. However, it was not significant (p > 0.05) for the unencapsulated biopeptides.A sustained significant decrease  were purchased from Sigma-Aldrich, Co. . Tween 80 was purchased from Merk Schuchardt OHG . All other reagents and chemicals used were of analytical grade and were obtained from Merk KGaA  and Sigma-Aldrich, Co. .Then, 10–15 week old male spontaneously hypertensive rats (SHRs) weighing between 250 to 320 g were obtained from the Animal Experimental Unit (AEU) Services at the Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia. The proposed animal utilization protocol was approved by the institutional Animal Care and Use Committee, Universiti Putra Malaysia .g. The supernatant was then lyophilized to obtain the ACE-inhibitory biopeptides with molecular weight (mw < 10 kDa) and stored at −40 °C before analysis.The AntBiop were prepared as previously described by Auwal et al. ,38. Briew/v. The surfactant, tween 80 was also prepared at 0.5% v/v and added to the solution of the AntBiop at a volume ratio of 4:1. The AntBiop-tween 80 mixture was then transferred to the stirring solution of chitosan at a chitosan:AntBiop mass ratio of 4:1 and sheared in an IKA® T 25 digital ULTRA-TURRAX® high shear homogenizer  for 15 min. The cross-linking agent, sodium tripolyphosphate, prepared at 0.5% w/v, was then added drop-by-drop to the above solution under continuous stirring for another 15 min. The unencapsulated biopeptides (mw ˂ 10 kDa) were removed by centrifugation at 10,000× g for 10 min. The resulting Chit-AntBiop-NPs were ice-bath sonicated for 10 min and filtered through a 0.45 µm membrane filter before storage at 4 °C. The blank chitosan nanoparticles were similarly prepared to serve as a control.The Chit-AntBiop-NPs were produced by the ionotropic gelation method under the optimum condition of AntBiop:chitosan mass ratio, stirring speed (rpm) and stirring time (min). Briefly, the AntBiop and chitosan were both prepared at a concentration of 0.5% ζ were measured at 25 °C in a Zetasizer . The Chit-AntBiop-NPs suspension was diluted in deionized water and the measurements were recorded as mean ± standard deviation of five experiments.The average particle size, pdi and The amount of the biopeptides entrapped within the matrix of the chitosan nanocapsules was determined as follows. The unencapsulated AntBiop in the supernatant was quantified using a Bichinchoninic acid micro protein assay kit obtained from Sigma-Aldrich, Spruce St., St. Louis, MO, USA. The amount of the entrapped AntBiop was then determined from the total amount initially added according to the following equation:The physical appearance of the Chit-AntBiop-NPs was studied by transmission electron microscopy . The samples were prepared by dilution with deionized water. After negative staining with 1% uranyl acetate solution for 1 min 30 s, a thin film of the sample was then mounted on to a grid of copper electron microscopy for visualization.Both the control and Chit-AntBiop-NPs were freeze dried and their thermal behavior was examined by differential scanning calorimetry using a digital scan colorimeter DSC instrument . Each sample was weighed into an aluminum pan and hermetically sealed. An empty pan was used as a reference and the thermograms were recorded within a temperature range of 40–200 °C at the heating rate of 10 °C/min.Tf − To, was calculated from the values of the end temperature (Tf) and onset temperature (To).The temperature difference E0 stands for the entrapment efficiency at a time t = 0 before the start of dialysis and iE is the entrapment efficiency at a time t = i (where i is 1 to 12 h) during the course of the dialysis.The in vitro release profile of AntBiop from Chit-AntBiop-NPs was monitored by dialysis in a 12–14 kDa molecular weight cut-off (MWCO) dialysis tube against phosphate buffered saline pH 7.4. The Chit-AntBiop-NPs and the control capsule were both transferred in to the dialysis tube and suspended in the release media at 37 °C with shaking at 250 rpm for 12 h. Then, 1 mL of the Chit-AntBiop-NPs was withdrawn at 1 h interval and the amount of the released AntBiop was measured as in The in vivo antihypertensive efficacy of the Chit-AntBiop-NPs was evaluated on SHRs. The study involved a total of 36 rats which were assigned into six groups of six rats each. They were acclimatized for one week in a controlled temperature room (22 °C) with a 12 h light/dark cycle and allowed free access to water and a standard commercial laboratory diet . The rats in each group were orally administered once with single dose by oral gavage. Group I (negative control) received blank Chit-NPs , Group II (positive control) were given captopril , group III were administered with unencapsulated biopeptides/AntBiop  while groups; IV, V and VI were treated with varying doses of Chit-AntBiop-NPs . The blank chitosan nanoparticles and Chit-AntBiop-NPs were dissolved in normal saline and 0.1 N HCl before administration. The volume of each dose given was determined based on body weight of the SHRs.The rats were placed in a holder, then pre-warmed to 35 °C on a heating flat form and the blood pressure was measured at 0 h before administration and then at 2, 4, 6, 8 and 24 h after administration to evaluate the SBP-lowering effect due to each dose using a non-invasive tail cuff device .p <0.05 was taken to be statistically significant.The statistical analysis was carried out using Minitab 16.0 software . Data analysis was carried out using one-way analysis of variance (ANOVA). A difference at p < 0.05), dose-dependent and sustained blood-pressure-lowering effect of the Chitosan-AntBiop-NPs, which was prolonged up to 24 h. Thus, the chitosan coating improved the gastrointestinal stability and bioavailability of the AntBiop. This demonstrated the relevance of the AntBiop-Chitosan-NPs for the management of hypertension.The Chit-AntBiop-NPs were fabricated under the optimum condition of AntBiop:Chitosan mass ratio, stirring speed and stirring time. The optimized capsules showed homogenously dispersed and small-sized particles of 162.47 nm with a high entrapment efficiency of 75.36%. The biopeptide- containing capsules showed a higher melting temperature of 146.78 °C compared to 137.59 °C for the blank capsules, with a cumulative release of 58% after 12 h in PBS pH 7.4. Furthermore, the in vivo antihypertensive efficacy indicated a significant (


Mammalian sleep alternates between sensory-responsive and protected offline periods via coordinated neural and cardiac rhythms. Rodents sleep in bouts lasting minutes; humans sleep for hours. What are the universal needs served by sleep given such variability? In sleeping mice and humans, through monitoring neural and cardiac activity , we find a previously unrecognized hallmark of sleep that balances two fundamental yet opposing needs: to maintain sensory reactivity to the environment while promoting recovery and memory consolidation. Coordinated 0.02-Hz oscillations of the sleep spindle band, hippocampal ripple activity, and heart rate sequentially divide non–rapid eye movement (non-REM) sleep into offline phases and phases of high susceptibility to external stimulation. A noise stimulus chosen such that sleeping mice woke up or slept through at comparable rates revealed that offline periods correspond to raising, whereas fragility periods correspond to declining portions of the 0.02-Hz oscillation in spindle activity. Oscillations were present throughout non-REM sleep in mice, yet confined to light non-REM sleep (stage 2) in humans. In both species, the 0.02-Hz oscillation predominated over posterior cortex. The strength of the 0.02-Hz oscillation predicted superior memory recall after sleep in a declarative memory task in humans. These oscillations point to a conserved function of mammalian non-REM sleep that cycles between environmental alertness and internal memory processing in 20- to 25-s intervals. Perturbed 0.02-Hz oscillations may cause memory impairment and ill-timed arousals in sleep disorders. All mammals benefit from sleep in fundamental aspects for brain and body . During this period, non-REM sleep occurred in bouts ranging from 8 to >512 s in duration, with a mean length of 108.6 ± 8.1 s. Both sigma power and SWA were elevated during non-REM sleep and decreased during waking or REM sleep . These combined results demonstrate that mouse non-REM sleep contains a 0.02-Hz oscillation of sigma power dynamics, corresponding to a periodicity of ~50 s.To examine whether mouse non-REM sleep shows microarchitectural dynamics indicative of variable fragility, we used polysomnography [electroencephalography (EEG)/electrocorticography (ECoG) and electromyography (EMG)] in freely moving mice , corresponding to a cycle length of 52.6 ± 2.6 s, comparable to mice. The sigma power band showed the most pronounced dynamics of around 0.02 Hz, while adjacent frequency bands displayed distinctly weaker periodicity . Together, these data unravel a 0.02-Hz oscillation common to both human and mouse non-REM sleep that is prevalent for sigma power and most prominent for fast spindles in human non-REM sleep.To explore whether this infraslow 0.02-Hz oscillation exists in higher mammals, we carried out a comparable power analysis for human sleep (fig. S5). As expected, sigma power was high during stage 2 (S2) sleep (light sleep) and declined during slow-wave sleep  , G and Hn = 6). Under these recordings conditions, the three major vigilance states wake, non-REM, and REM sleep showed spectral profiles comparable to those of freely moving animals (fig. S8). The 0.02-Hz oscillation in sigma power of non-REM sleep was present in the simultaneously recorded EEG and LFP signals  analysis of variance (ANOVA) for factors “frequency” and “area”; F1,5 = 145.8, P = 6.88 × 10−5; F4,20 = 19.23, P = 1.25 × 10−6; n = 6; paired t test, t = 17.88, P = 1.01 × 10−5 for SI; t = 5.72, P = 0.0023 for SII; n = 6; paired t test, t = 2.83, P = 0.037 for AC; t = 2.02, P = 0.1 for mPFC).To assess whether 0.02-Hz oscillations were present in local cortical circuits, we performed multisite referential local field potential (LFP) recordings across four cortical areas in combination with polysomnography in sleeping head-fixed mice , as assessed by polysomnography , suggesting a phase advancement for early over late arousals. The progression into the declining sigma power period thus reflects the entry into a period of sleep fragility. Last, we asked whether the total duration of the non-REM sleep before noise exposure affected responsiveness. Both wake-up and sleep-through trials were broadly distributed across the range of non-REM sleep bout durations in mice (fig. S10), ruling out bout duration as a determinant of behavioral outcome to noise exposure.If 0.02-Hz oscillations are relevant for sleep fragility and continuity, then they should be accompanied by a varying arousability of mice in response to external stimuli. We chose acoustic stimuli such that they lasted half a cycle of the 0.02-Hz oscillation (20 s). This long-duration noise would probe the propensity to arouse over the sustained periods of low and high sigma power and hence reveal whether these corresponded to states of distinct fragility. A white noise stimulus of 90-dB sound pressure level (SPL) yielded an arousal success rate of 38.7 ± 8.6% . Thus, hippocampal ripple activity was high during periods of increasing sigma power, during which mice maintained sleep while being exposed to noise. This suggests that more pronounced 0.02-Hz oscillations strengthen offline consolidation of hippocampus-dependent memory.We further characterized the 0.02-Hz oscillation by examining the timing of ripples (150 to 250 Hz) in the CA1 area of the hippocampus, which represent an established index for offline memory processing . The 0.02-Hz oscillation peak also correlated with the mean fast spindle density . Spindle measures per se (such as spindle density and spindle count) in this analysis were not significantly correlated with episodic memory recall . These data are the first indications that the periodic clustering of spindle activity on a 50-s time scale is a critical determinant for offline memory consolidation.To explore the role of 0.02-Hz oscillations in memory consolidation, we correlated the explicit postsleep recall on an episodic memory task (presented before sleep) in humans with oscillation peaks and conventional measures of spindle density , we found that heart rate increased and fluctuated around elevated values when sigma power oscillations were declining , A and BSleep has to reconcile the needs for continuity and fragility. Here, we uncovered a 0.02-Hz oscillation in mouse and human non-REM sleep with characteristics that qualify it as a hallmark for how sleep balances these conflicting needs. First, the 0.02-Hz oscillation is most prominent in a frequency band that contains neural rhythms associated with the gating of sensory information during sleep. Second, it is coordinated with an established physiological correlate of offline memory processing and with modulation of autonomic status. Third, the 0.02-Hz oscillation phase is linked to wake-up from sleep in mice and to the extent of overnight memory consolidation in humans. This dual behavioral relevance in two different species suggests that the 0.02-Hz oscillation provides a unitary temporal scale of mammalian non-REM sleep over which both beneficial effects and maintained reactivity to the environment are balanced.The 10- to 15-Hz frequency band analyzed here contains sleep spindles, a well-described sleep rhythm that is a thalamocortically generated and visually obvious hallmark of the non-REM sleep EEG in humans and carnivores between ~8 and 16 Hz – and high-frequency (10 to 30 Hz)–dominated states in cycles of 60 to 80 s  that is prominent in sleep disorder patients, consisting of visually identifiable alternations in EEG synchrony over periods of 10 to 60 s . Food and water were administered ad libitum. Surgery for combined EEG (ECoG)/EMG electrode implantation was performed on a total of 26 5- to 7-week-old male C57BL/6J mice, bred in our colonies, as previously described by Wimmer 2 and N2O mixture), and two gold-plated screws (1.1-mm diameter) (2 and N2O mixture) on eight mice (For EEG (ECoG)/EMG surgeries, mice were anesthetized with isoflurane /EMG signals were recorded in freely moving mice, acquired and amplified using an Embla amplifier (gain 2000×), digitized at 2 kHz, and down-sampled to 200 Hz using Somnologica version 3.3.1 software (Embla System). The EEG (ECoG) and EMG traces were high-pass–filtered at 0.7 and 10 Hz, respectively. A 48-hour baseline sleep-wake recording under undisturbed conditions was obtained for every animal, and only the 100 min after light onset for the two consecutive days was used for further analysis to assess a data set homogeneous with respect to time of day. Recordings of LFPs were obtained from head-fixed mice habituated to sleep (fig. S8). The EEG (ECoG)/EMG signals allowed to assess the behavioral state during the sleep-wake cycle recordings and, together with LFP signals, were amplified (1000×) and acquired through Plexon Systems (16-channel Multiple Acquisition Processor system). More specifically, the signals were sampled at 1 kHz, high-pass–filtered at 0.8 Hz, and low-pass–filtered at 300 Hz. LFP electrode positions were labeled at the end of all recordings through electrocoagulation before transcardiac perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer , through current injections , and post hoc compared to the stereotactic atlas after coronal slicing (100-μm sections) (fig. S8). Mice with an unprecise electrode localization were excluded from specific analyses. Head-fixed mice were not exposed to noise stimuli.et al. . The memory study also included standard polysomnographic full-night EEG recordings with a higher density of electrode sites.Human data obtained from 27 healthy men  who participated in a previous pharmacological study included overnight polysomnographic and electrocardiographic (ECG) recordings , EMG , and electrooculography (around the eyes), with the memory study data set using additional EEG sites . Electrode impedances were kept below 5 kilohms. Signals were amplified , digitized (sampling rate >200 Hz), and filtered (EEG and electrooculogram between 0.3 and 35 Hz and EMG between 10 and 100 Hz).Polysomnographic recordings included EEG from C3 channel mainly used for sleep scoring in the core study, whereas analyses in the memory group used all nine recording sites. Data of subsequent analyses were down-sampled to 100 Hz to facilitate computation. Analysis was performed in MATLAB 2013b (MathWorks) using custom-made scripts, FieldTrip (www.ru.nl/neuroimaging/fieldtrip) (www.spisop.org). For the correlation of the standard sleep parameters with memory and with the strength of the 0.02-Hz oscillation, the average of all parietocentral EEG electrodes  was taken for the spindle band analyses, and the average of the frontal electrodes  was taken for the SWA band analysis, because these locations correspond to the sites with the highest overall power in the respective bands.Sleep stages were scored offline in 30-s epochs by an experienced scorer according to standard criteria . Mean power density from the spectra was calculated in the frequency band for slow waves (0.5 to 4 Hz) and in the sigma band (10 to 15 Hz). Concrete fast spindles and slow waves during non-REM sleep were analyzed according to previously published algorithms  using a digital finite impulse response (FIR) filter . Next, all intervals of time with consecutive positive-to-negative zero crossings were marked as putative slow waves if their durations corresponded to a frequency between 0.5 and 1.11 Hz  with a ±1-Hz range (−3 dB cutoff). Then, using a sliding window with a size of 0.2 s, the root mean square (RMS) was computed, and the resulting signal was smoothed in the same window with a moving average. A spindle was detected when the smoothed RMS signal exceeded an individual amplitude threshold by 1.75 × SD of the filtered signal in this channel at least once and additionally exceeded a lower threshold of 1.5 × SD for 0.5 to 3 s. The crossings of the lower threshold marked the beginning and end of each spindle and quantified their length. Spindle amplitude was defined by the voltage difference between the largest trough and the largest peak. Spindles were excluded for amplitudes >200 μV.The scheme in fig. S2 summarizes the analysis steps for undisturbed non-REM sleep in mice. From the scored data, all non-REM sleep bouts ≥24 epochs  were selected in the first 100 min at ZT0 for two consecutive light phases, regardless of the amount of non-REM sleep (fig. S2A). The spectral files of all 4-s epoch across all non-REM sleep (red lines in hypnogram in fig. S2A) were then used to calculate an arithmetic mean FFT per mouse. In recordings from head-fixed sleeping mice, an average non-REM sleep spectral profile was calculated across the entire recording for each mouse (810 to 3210 s).Figure S2B shows how the time course of spectral power was analyzed in 4-s bins for the following frequency bands: SWA (0.75 to 4 Hz), theta (6 to 10 Hz), sigma (10 to 15 Hz), and beta (16 to 20 Hz). To do this, the power values from the 4-s FFTs for each frequency band were normalized to the average non-REM sleep FFT calculated across all non-REM sleep (fig. S2A) and plotted against time, yielding the line graphs in The spectral profiles of these power time courses of each non-REM sleep bout were obtained through calculating an FFT (fig. S2C) with Hamming window method, which revealed all power values in the infraslow frequency range (<0.125 Hz). The choice of a minimal bout length of 24 epochs, corresponding to ≥96 s, preserved at least two cycles of the 0.02-Hz oscillation detected here. A mean spectral profile was calculated for every mouse through averaging across the interpolated absolute FFTs obtained from each power profile of each non-REM sleep bout .f) with equal bout length distribution did not yield a peak unless a 0.02-Hz sine wave was added . Before FFT calculation, the average power value for each frequency band was subtracted from each non-REM sleep bout to prevent large power increases at extremely small frequencies; however, the 0.02-Hz peaks were also present without this offset. Autocorrelations were calculated for original and shuffled sigma power data of these long non-REM bouts for each mouse .Control analyses were carried out to ensure the robustness of peak detection (as shown in fig. S3). For the analysis of scale-free behavior, similar FFT calculations for a simulated scale-free power profile /LFP recordings, the FFTs over the power data mentioned above were obtained for each non-REM sleep bout using a Hamming window and means calculated as before . LeavingLast, to determine peak and SDs of the FFTs, we performed a Gaussian fit (one term). Peak location and SD values were obtained from the fitted curve and used to calculate the average peak value in the range (peak ± 0.5 × SD). These values were used to calculate average peak values in all other frequency bands . By chooThe peak ± SD frequencies of the FFT for sigma power were also used to calculate a band-pass (FIR)–filtered trace of sigma power time course before noise onset  and to rn = 1 of 10 animals did not wake up in any of the noise exposures and did not contribute to the power calculations . For any successful trial, the values in both sigma and SWA frequency bands were expressed in percentage with respect to the average non-REM sleep power spectrum in the 40-s prestimulus period , C and Eulations , D and ERipple activity time course was quantified from the LFP recording in the CA1 area. The signal was first filtered between 150 and 250 Hz and then squared. The values were averaged in a 4-s bin, and a cross-correlation was performed against the corresponding sigma power from channel SI (resampled at 1 Hz) using the ripple activity trace as “source wave.”The scheme in fig. S5 summarizes the analysis steps for human non-REM sleep. An analysis similar to that in mice was performed on consecutive 30-s intervals of non-REM sleep EEG (further referred to as bouts) of the first 210 min of sleep and free of artifacts or movement arousals for the core study data set (fig. S5A) and was extended to the full-night sleep in the memory group data set  and 5. TAfter visual confirmation that the power time course in the sigma and SWA bands corresponded to real spindle and SWA activity, the temporal resolution was reduced to highlight activity changes in the infraslow periodicity. Therefore, a symmetric moving average was applied in a 4-s time window to match the resolution of the mouse data. As in mice, the average absolute values of the non-REM sleep spectral composition were used for normalization by setting them to 100% , E and FTo establish the spectral profile of these power time courses, the spectra of the power time courses for each frequency band were obtained for all non-REM sleep bouts lasting ≥120 s (≥4 epochs) . For this, a Morlet wavelet analysis was performed in each interval on the previously calculated power time courses for respective frequency bands that aimed for a frequency resolution of 0.001 Hz in the range 0.001 to 0.12 Hz with time steps of 0.5 s. To obtain one power spectrum for each bout, we then averaged the resulting signal across time steps along the duration of the bout (fig. S5D).Last, the spectral profile of the 0.02-Hz oscillation of all frequency bands for all non-REM sleep was calculated by averaging the spectral values across all the non-REM sleep bouts of the subject, weighted by their duration. As in mice, the spectral profile obtained per subject was normalized to its own mean. To determine peak and SDs of each power spectrum, a Gaussian fit (three terms) was performed on the normalized power from single subjects. The peak location and SD values were obtained from the fitted curve and used to calculate the average peak value in the range (peak ± 0.5 × SD) , G and HTo address specificity of the 0.02-Hz oscillation to spindles, we repeated the analysis for a frequency band tailored to the individual spindle band of each subject. Thus, for each subject, the frequency band was centered to its FSP that was determined according to previously described standard methods  or triple the length (≥360 s), changing Morlet wavelet cycle length to seven cycles for better frequency resolution, skipping the normalization steps, or comparing the power fluctuations in the full spectrum  essentially yielded the same results reported here. Autocorrelations were calculated as in mice for original and shuffled sigma power profile data of non-REM sleep. To more closely match the respective analyses in mice, the data were split in all possible continuous 240-s segments and down-sampled to 1 Hz .A subset of 10 mice implanted for polysomnography was habituated to the experimental noise stimulus during the period of tethering after surgery through playing noises six to eight times per light phase. During the experimental trials, animals were exposed, four at a time, to white noise pulses of 90-dB SPL lasting 20 s, generated through custom-written LabVIEW procedure . The duration of the noise was chosen such that it covered half a cycle of the 0.02-Hz oscillation and because mice woke up or slept through it at comparable rates. The arousal success rate was defined as the fraction of wake-up trials within all accepted trials. In a preliminary experimental series, a 20-s pulse at 80 dB was found to lead to an insufficient number of wake-up trials, with the arousal success rate <30%, whereas a 4-s pulse at 100 dB led to arousals in more than 90% of the cases. Noise was played randomly in the first 100 min at ZT0, but the following conditions had to be fulfilled: At least one of the four mice was in non-REM sleep for ≥40 s, as assessed through online monitoring of EEG (ECoG)/EMG traces, and the previous noise had been played ≥4 min before. The experimenter was blind to the spectral composition of non-REM sleep during the noise exposure experiment, such that noises were played without knowledge of the sigma power phase. Wake-ups were identified on the basis of characteristic alterations of EEG/EMG signals, namely, the decrease in amplitude and increase in frequency for the EEG trace, combined with the detection of muscular activity from the EMG electrodes . The aniIn the core study, subjects arrived at the laboratory at 9:00 p.m. for experimental preparation, and sleep was allowed between 11:00 p.m. (lights off) and 7:00 a.m. Subjects underwent blood sampling via an intravenous forearm catheter, which was connected to a long thin tube and enabled blood collection from an adjacent room without disturbing the subject’s sleep and unnoticed by all subjects  whether a face was new or presented during one of the task episodes, and for the latter, whether it occurred in the first or second episode and at which location it occurred. Episodic memory, that is, “what-where-when” memory, was determined by the percentage of the faces that were correctly identified as occurring in one of the episodes , and for which the participant also correctly indicated the episode  and the location  it occurred, minus the locations for which the participant had forgotten that they were occupied with any face in a final separate recall test .n = 12 mice (eight freely moving and four head-fixed), the heart rate was quantified through detection of R waves in the EMG trace filtered between 20 and 300 Hz. Reliability of this signal was confirmed through standard ECG recording in one mouse (fig. S12) . In both mice and humans, the cross-correlation was performed using the heart rate signal as source wave. In both species, the cross-correlograms were first averaged within and then across subjects. In humans, all of the above procedures were repeated separately for S2 and SWS epochs instead of non-REM sleep epochs but reported solely for the oscillation peak analysis.For cross-correlating time courses of heart rate (in beats per minute) and sigma power in mice, the sigma power trace was interpolated to match the 1-Hz sampling rate of the heart rate trace. In humans, it was smoothed in a 4-s moving symmetric time window, and both sigma power and heart rate traces were resampled at 100 Hz by interpolation. The 120-s intervals were W test, and parametric or nonparametric statistical tests were chosen accordingly. The Wilcoxon signed-rank test was used for nonparametric matched pair comparisons, whereas paired two-tailed Student’s t tests (referred to as t test in text and legends) were used as parametric statistical tests. RM ANOVA was used as parametric statistical test to study the within-subjects effect of behavioral outcome and/or the between-subjects effect. To assess equality of variances for the RM ANOVA, we calculated the Mauchly’s test of sphericity. Whenever equality of variances was rejected, the univariate adjusted Greenhouse-Geisser correction was applied. Friedman rank sum test was used as a nonparametric test to study within-subjects effect in case parametric model parameters of an ANOVA violated assumptions of normality. Statistical tests were calculated using JMP version 10.0.0 (SAS Institute Inc.), Igor Pro, and the R programming language  . P < 0.05 was considered statistically significant. If not mentioned otherwise, P values were reported uncorrected for multiple comparisons, as taking these into account did not alter the main results. For the purpose of comparison between mice and humans, all data in bar graphs are presented as means ± SEM, even if not normally distributed (n refer to either mice or humans. Time course graphs, as well as data in the main text, are presented as means ± SEM.Group size in mice matched the minimum required to obtain a statistical power of 0.8. Power analyses were carried out on the basis of effect sizes obtained from preliminary data sets. Group size in humans was chosen to obtain a statistical power of 0.95 for similar effect sizes, as observed in mice. Statistical power was calculated using G*Power version 3.1.9.2. Data normality was tested using the Shapiro-Wilk


Listeria innocua ATCC 33090 was investigated. The USP was up-regulated not only in the stationary phase but also during the exponential growth phase. The three dimensional (3D) structure of USP was predicted using a combined proteomic and bioinformatics approach. Phylogenetic analysis showed that the USP from Listeria detected in our study was distant from the USPs of other bacteria  and clustered in a separate and heterogeneous class including several USPs from Listeria spp. and Lactobacillus spp. An important information on the studied USP was obtained from the 3D-structure established through the homology modeling procedure. In detail, the Model_USP-691 suggested that the investigated USP had a homo-tetrameric quaternary structure. Each monomer presented an architecture analogous to the Rossmann-like α/β-fold with five parallel β-strands, and four α-helices. The analysis of monomer-monomer interfaces and quality of the structure alignments confirmed the model reliability. In fact, the structurally and sequentially conserved hydrophobic residues of the β-strand 5 (in particular the residues V146 and V148) were involved in the inter-chains contact. Moreover, the highly conserved residues I139 and H141 in the region α4 were involved in the dimer association and functioned as hot spots into monomer–monomer interface assembly. The hypothetical assembly of dimers was also supported by the large interface area and by the negative value of solvation free energy gain upon interface interaction. Finally, the structurally conserved ATP-binding motif G-2X-G-9X-G(S/T-N) suggested for a putative role of ATP in stabilizing the tetrameric assembly of the USP. Therefore, the results obtained from a multiple approach, consisting in the application of kinetic, proteomic, phylogenetic and modeling analyses, suggest that Listeria USP could be considered a new type of ATP-binding USP involved in the response to acid stress condition during the exponential growth phase.An Universal Stress Protein (USP) expressed under acid stress condition by  Listeria innocua is regarded as a non-pathogenic indicator for the presence of Listeria monocytogenes in foods  and the glutamate decarboxylase (GAD) pathways  at 37°C and then stored in cryovials  at −80°C. Prior to use, cells were propagated twice in the same medium and incubation conditions, and collected in the middle of exponential phase. The growth was assessed for 30 h in 500 mL of BHI  and in BHI adjusted at pH 4.5 . Moreover, a further trial was performed using pre-acid-adapted cells. For this purpose, cells cultured under acid stress condition were collected in the middle of exponential phase and inoculated in BHI (batch pa-C) and in BHI at pH 4.5 (batch pa-AS). In all cases an initial inoculum of about 107 CFU/mL was used.Plate counts were performed in BHI agar at different intervals and the growth kinetic parameters were estimated with the D-model of Baranyi and Roberts  using thL. innocua ATCC 33090 cultivated as described above  were collected by centrifugation  in the middle of the exponential phase and during the stationary phase. Cells were washed three times with Tris-HCl , standardized at an OD600-value of 1.0 and re-suspended in a lysis buffer . Eight glass beads (Ø 0.4 mm) were added to each cellular suspension (140 μL), then suspensions were vortexed (3 min), incubated for 2 h at 37°C, vortexed (3 min), and sonicated for 5 min with an ultrasonic homogenizer  using a probe of 0.5 mm diameter. After centrifugation at 17,500 rcf for 30 min at 4°C, the pellet was discarded and the supernatant (lysis buffer), containing the protein extract, was subjected to the Bradford-based protein assay kit  to determine the protein concentration. Bovine serum albumin was used as a standard.Cells of L. innocua ATCC 33090, cultivated as above , were collected from the lysis buffer with methanol/chloroform according to the method described by Wessel and Flügge . Spot detection, quantification, and analysis, were performed using the PDQuest™ 2-D gel analysis software, Version-8 .2 laser, acquiring at least 400 laser shots from each sample. A peptide mixture (Sigma-Aldrich Co.) was used as external standard. Proteins were identified with the MS-Fit Protein Prospector software (website: http://prospector.ucsf.edu), searching the Uniprot database (2015.3.5 vers.). C-carbamidomethylation was set as a fix modification, while pyroglutamic acid at N-terminal glutamine and methionine oxidation were variable modifications. Up to one trypsin missed cleavage was allowed and peptide mass tolerance was 40 ppm. Search was taxonomically limited to microorganisms and then refined, with taxonomical restriction to Listeria spp. Only protein hits with at least 15 matching peptides, MOWSE score higher than 108, and coverage higher than 25% were considered as positive identifications. Protein identification was validated by a software-assisted comparison of experimental and expected Mw and pI, as inferred from the UniProtKB/Swis-Prot database through the TagIdent tool (http://web.expasy.org/tagident/).Proteins with modified expression level were identified by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS)-based peptide mass fingerprinting (PMF). Briefly, protein spots were manually excised, destained, and digested overnight at 37°C with 12 ng/mL proteomic grade trypsin . Afterwards, peptides were extracted in 1% formic acid/acetonitrile (1:1), vacuum dried, and analyzed using α-cyano-4-hydroxycinnamic acid (10 mg/mL in 50% acetonitrile/0.1 TFA) as the matrix. Mass spectra were obtained in the reflectron positive ion mode on a MALDI-TOF Voyager-DE™ PRO mass spectrometer (Applied Biosystems) equipped with a 337 nm NA0A0E1Y4Z4) from L. monocytogenes FSL F2-208 was used as target. Homolog sequences and the USP template sequence were searched in the non-redundant protein sequence database (NR) and Protein Data Bank (PDB) respectively, using the protein-protein Basic Local Alignment Search Tool (BLASTp). The USP structure (PDB ID: 3S3T) from Lactobacillus plantarum was chosen as template for the model building due to its protein sequence showing the highest alignment score . In detail, the four homolog chains A, B, C, and D of the complete USP structure from Lb. plantarum were aligned with the target sequence of Listeria  of the USP . In fact, the non-pre-acid-adapted cells  than that exhibited in C (μmax of about 0.14 h−1). Moreover, non-pre-acid-adapted cells cultured in conventional conditions (C) reached the stationary phase (yend ≈1.99 CFU/mL) in about 12 h, whereas in acid stress conditions (AS) the maximum population (yend ≈5.28 CFU/mL) was reached in about 20 h  between pa-C and pa-AS were found in the final microbial levels  and acid stress (AS) conditions on the growth kinetics of non-pre-acid-adapted cells of s Figure  showed aL. innocua ATCC 33090 in the exponential growth phase and the proteome of cells cultivated in conventional  and acid stress  conditions was compared by 2-DE. More than 500 spots were detected in each gel apart from the cultural conditions. The image analysis highlighted significant differences between conventional and acidic stress conditions. In detail, an up-regulation of several spot proteins was detected in the proteome of cells cultivated in acid stress conditions . In detail, after normalization over a number of protein spots with unmodified expression, densitometric analysis of the spot intensity evidenced 19 differentially expressed gene products at a threshold ≥2 (p < 0.05), which were selected for the MS-based identification  but the primary structure was identical to the USP from L. monocytogenes (Uniprot accession number A0A0E1Y4Z4), sharing each other 100% of structural homology  were highly up-regulated, with expression ratio ranging from 3 to 8, namely succinate-semialdehyde dehydrogenase, NADP-dependent aryl-alcohol dehydrogenase, general stress protein (CTC), and (USP). Using the MS-based approach, the USP was identified in the proteome of gy Table .L. innocua in different conditions . Similar sequences were searched into non-redundant protein sequences database using BLASTp. About 100 protein sequences, with 140–161 residues, showed a high sequence identity (56–100%) with a score ranging from 181 to 314 and a E-value from 9e−55 to 6e−107. The multi-sequences alignment .USPs proteins are highly conserved and all deemed important for the stress response. The FASTA sequence (160 aa) of USP from t Figure  showed ht Figure . Moreoveidentity 6–100% wit Figure , UspA, Un et al.  and formLb. plantarum and L. monocytogenes strains, such as USP EHS84548 USP1 and USP Q8Y6V1_LSMO, involved in acid stress resistance.This last class, in addition to the template and target sequences, includes also USPs from L. innocua ATCC 33090 were stored as PDB output file and the best model (Model_USP-691) with a lower DOPE score (−0.247) was refined and used for both validation and interface analysis. Model_USP-691  and the template 3s3t (green) from Lb. plantarum was, as expected, very close, and small differences were observed in the N-terminal and C-terminal of helix α2.The Model_USP-691 showed a good accuracy in both the stereochemical properties and the absolute quality of the structure. The main chain-conformations for 95.9% aminoacid residues were allocated within the most favored region of Ramachandran plot, only two residues (GLN 169B and ASN 378C) were found in disallowed regions of the plot Figure . The rel2 (PISA) or 1000 Å2 (POPSCOMP) involving a total of ~25% (PISA) or ~30% (POPSCOMP) of residues (Table 2 (PISA) or 800 Å2 (POPSCOMP) including the 20% (PISA) or 22% (POPSCOMP) of residues  was compared with the 3D structures of USPs available in the PDB by means of the DALI server Table . Results2 Figure . The regL. innocua ATCC 33090 was used as a surrogate of the pathogen species L. monocytogenes, to investigate the effect of sub-lethal acid pH on the growth and protein expression. In agreement with results obtained for the pathogenic microorganism .In fact, the results highlighted that under acid stress conditions, 19 gene products were at least two-fold up-regulated in 2. The GABA shunt can operate as an alternative pathway to provide succinate in some steps of the tricarboxylic acid (TCA) cycle in bacteria, including L. monocytogenes, that lacks of a complete set of genes necessary for the TCA cycle  shunt pathway  clustered in a separate and heterogeneous class, arbitrarily called USPL. Therefore, we can assume that the USP from the USPL class could play a different role in the stress response than USPs grouped in other classes.Herein, USP over-expression was associated with the cellular growth arrest of 146-L147-V148-V149) of the β-strand 5 seem to have an essential role into monomer-monomer interface formation  as well, but only the residues I139 and H141 resulted conserved in the sequence. Probably these residues, together with the others found conserved in the structure and sequences of USPs, may function as hot spots in monomer–monomer interface assembly  between the chains A and B or C and D of model, compatible in size with the interfaces (740–1900 Å2) of other crystallographic resolved USPs. In addition, a complementary electrostatic charge distribution was found at the interface. Moreover, the negative value of solvation free energy gain upon formation of the interface, as well as the high number of hydrogen bounds found at the interface, suggest a favorable interaction between the USP monomers. The proposed USP assembly . We found this motif present in the structure of Model_USP-691-A, and structurally conserved compared to the more closely related USPs  protein did not show any ATPase activity, but was able to bind ATP though lacking the specific ATP binding motif. Therefore, the biochemical and biological function of the USPs could rely on an ATP dependent-factor, which is likely to be linked to a specific energetic state of the cell.Structural and biochemical studies suggest a wide array of functions of USPs. Anyway, few USP structures are available in PDB and there is little structural information is available for most of them. We found that of the 20 proteins with a sequence similar (Z-score significance ≥15.0) to the Model_USP-691-A, only for about 50% of them there is available structural information Table . General Figures , 7, 9. Ir Figure , could pr Figure  found thy Figure  the USP y Figure . Recently Figure  showed tListeria USP might be deemed as a new type of ATP-binding USP. Contrarily to USP-types involved into growth arrests, the USP of L. innocua could have a key role in the response to acid stress during the exponential growth phase.In conclusion, based on the current structural prediction, PT: design of the work, analysis and interpretation of the microbial and proteomic data, drafting the work; MS: interpretation of the data, drafting the work, and revising it critically; RC: conception of the work, drafting the work, and revising it critically; ES: involved in experimental designing; LT: analysis of the microbial growth; GPi: analysis and interpretation of mass spectrometry data; GPa: design of the work, execution of 2-DE and bioinformatic analyses, interpretation of data, drafting and revising the work. FF: design of the bioinformatics approaches, agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Autoantibodies directed against cytosolic 5′-nucleotidase 1A have been identified in many patients with inclusion body myositis. This retrospective study investigated the association between anticytosolic 5′-nucleotidase 1A antibody status and clinical, serological and histopathological features to explore the utility of this antibody to identify inclusion body myositis subgroups and to predict prognosis.Data from various European inclusion body myositis registries were pooled. Anticytosolic 5′-nucleotidase 1A status was determined by an established ELISA technique. Cases were stratified according to antibody status and comparisons made. Survival and mobility aid requirement analyses were performed using Kaplan-Meier curves and Cox proportional hazards regression.Data from 311 patients were available for analysis; 102 (33%) had anticytosolic 5′-nucleotidase 1A antibodies. Antibody-positive patients had a higher adjusted mortality risk , lower frequency of proximal upper limb weakness at disease onset  and an increased prevalence of excess of cytochrome oxidase deficient fibres on muscle biopsy analysis , compared with antibody-negative patients.Differences were observed in clinical and histopathological features between anticytosolic 5′-nucleotidase 1A antibody positive and negative patients with inclusion body myositis, and antibody-positive patients had a higher adjusted mortality risk. Stratification of inclusion body myositis by anticytosolic 5′-nucleotidase 1A antibody status may be useful, potentially highlighting a distinct inclusion body myositis subtype with a more severe phenotype. Inclusion body myositis (IBM) is an acquired muscle disease that most commonly affects males aged over 45 years. Along with polymyositis (PM) and dermatomyositis (DM), IBM is usually classified as one of the idiopathic inflammatory myopathies. However, IBM differs in comparison with PM and DM, as sustained responses to immunosuppression are not seen, and histologically it is associated with significant degenerative features.6–8et al and the European Neuromuscular Centre (ENMC) criteria).13–15The diagnosis of IBM relies upon a combination of clinical and laboratory findings as defined in various diagnostic criteria , Griggs In IBM, autoantibodies directed against cytosolic 5′-nucleotidase 1A (cN-1A) have recently been identified. It is suggested that these may support the diagnostic process, as well as potentially providing clues as to disease pathogenesis.Pooled IBM case data from four European countries were used. Researchers based in Nijmegen, The Netherlands, coordinated data collection from The Netherlands, France and Sweden. Data collection in the UK was coordinated by researchers based in Manchester, UK.et al (‘definite’ or ‘possible’) or ENMC  diagnostic criteria for IBM and had sera available for anti-cN-1A antibody testing.11Included cases met either the MRC , Griggs Swedish, French and Dutch (‘non-UK’) patients were identified from clinical databases. Researchers blinded to anti-cN-1A antibody status  reviewed the medical records and retrospectively completed a standardised data collection pro forma. UK patients were identified from six centres contributing to the UKMYONET research study, coordinated by The University of Manchester. As part of this study, data are captured using a standardised pro forma at the time of study recruitment .10.1136/annrheumdis-2016-210282.supp1supplementary appendixData collected included demographic, clinical , laboratory findings (creatine kinase (CK) levels, muscle biopsy features, serological testing), comorbidity, mobility aid usage and mortality. In most cases, data were available regarding features present at disease onset and at the time of last patient review (or recruitment to the UKMYONET study in the case of the UK cohort). In all cases, ‘disease onset’ refers to the initial date that symptoms of IBM were noted, as reported by the patient. ‘Disease duration’ is defined as the period between disease onset and the date of anti-cN-1A antibody testing. Regarding mortality, in the non-UK cohort, the primary cause of death was categorised by review of the patient's medical records as either ‘respiratory’, ‘cardiac’, ‘cerebrovascular’, ‘malignancy’ or ‘other’. In the UK cohort, additional mortality and comorbidity statistics were obtained from the UK Health and Social Care Information Centre, including coded data regarding the cause of death where applicable. The cause of death in these cases was assessed and assigned to the same categories as the non-UK cohort.For all cases, the histopathology biopsy report performed at initial diagnostic interrogation was reviewed, and the presence of certain specific features determined from the report text. The reporting histopathologists were blinded to the anti-cN-1A antibody status of each patient at the time of reporting. Cytochrome oxidase (COX) deficient fibres in the biopsy sample were recorded as ‘excessive’ if the reporting histopathologist indicated that numbers were adjudged higher than expected, according to the patient’s age. In some cases, the date that the biopsy was performed was not available. In such instances, this was assumed to be the same as the date of diagnosis.All sera were analysed at the Department of Biomolecular Chemistry in Nijmegen by ELISA, with the three synthetic peptides containing cN-1A autoepitopes previously identified by overlapping peptide microarray analyses.22Data regarding the presence of myositis-specific antibodies (MSAs) and myositis-associated antibodies (MAAs) were collected where available. For the non-UK patients, data were obtained from results available in the medical records, and methodology of testing was unique to each centre. MSAs and MAAs in the whole UK cohort were screened by immunoprecipitation at the University of Bath  using previously described standardised methodology.The per-subject sum of all recorded comorbidities  and malignancy) was calculated. Current or previous smoking was also treated as a comorbidity for the purposes of this analysis. According to the number of these factors present, each patient was then assigned a comorbidity score of 0, 1 or 2 or more for use in regression. Differences in demographic features, comorbidities, clinical features, autoantibody status and muscle biopsy features between anti-cN-1A antibody positive and negative patients were assessed using logistic regression. In order to test the effect of potential confounders, adjusted (multivariable) logistic regression models were produced when unadjusted analysis had suggested a significant difference (defined as p<0.05).The impact of anti-cN-1A antibody status on survival and mobility aid requirement was assessed using Kaplan-Meier curves, log-rank testing and Cox proportional hazards regression modelling. In both cases, the start of the surveillance period was the date of disease onset. For the mobility aid analysis, subjects exited the model at the time of mobility aid requirement or at the time they were last known to have not required one. For the survival analysis, subjects exited the model at the time of death or at the time they were last known to have been alive. Each Cox regression model included adjustment for age of disease onset, gender and comorbidities. Other variables were added to the models if there was an a priori assumption that a relationship between anti-cN-1A antibody status and the outcome variable was likely to exist. For example, a higher incidence of anti-cN-1A antibodies in those with Sjögren's syndrome is reported, a more prominent bulbar involvement in anti-cN-1A positive patients with IBM has been described and a correlation between COX deficiency and more advanced age at biopsy could exist.The analysis plan specifically omitted correction for multiple testing due to the highly conservative nature of such methods which would risk elimination of potentially useful information which was sought to be retained, given the exploratory nature of this study. Data were processed and analysed using Stata for Windows V.13.0 . Kaplan-Meier curves were generated using GraphPad Prism V.6 (GraphPad Software).et al criteria) or ‘pathologically/clinically defined’ (for MRC criteria) IBM and anti-cN-1A antibody status was found . No difference was found in the interval between disease onset and the time of antibody testing between seropositive and seronegative groups (8.29 years (IQR 4.96–11.95) in the seropositive group vs 7.57 years (IQR 4.94–11.18) in the seronegative group, OR 1.01, 95% CI 0.97 to 1.06, p=0.604).After screening databases in the four involved countries, 311 patients meeting the study inclusion criteria were selected for further analysis . Overall, 33% (102/311) were positive for the anti-cN-1A antibody. No statistically significant differences were identified in demographic characteristics , CK levels, smoking history or comorbidities between the anti-cN-1A antibody positive and negative groups . Non-sigOf the whole cohort of 311 patients, 70 deaths were recorded (31/102 (30%) in the anti-cN-1A antibody positive group and 39/209 (19%) in the negative group). The mean age of death overall was 77.8 years (SD=8.2), with no significant difference detected according to anti-cN-1A antibody status (77.0 years (SD=7.7) in the seropositive group vs 78.4 years (SD=8.6) in the seronegative group, OR 0.98, 95% CI 0.92 to 1.04, p=0.482). The cause of death was known in 63% (44 of 70) of cases. An excess of deaths as a result of respiratory disease was evident in the anti-cN-1A antibody positive group (16/25 (64%) in the anti-cN-1A antibody positive group and 9/25 (36%) in the negative group, OR 4.23, 95% CI 1.79 to 9.97, p=0.001). Adjusted analysis was not performed here due to the low numbers available for analysis. Death from other causes  did not differ between anti-cN-1A antibody positive and negative groups.Data from 300 patients, where the date of disease onset and date of last follow-up (or date of death) were known, were available for further analysis. This included 66 of those that had died  and comprised a total of 3550 patient-years of follow-up. The median survival in the anti-cN-1A antibody positive group was 17.6 years compared with 24.2 years in the antibody-negative group, and the Kaplan-Meier curves were significantly different . After adjustment for age at disease onset, gender and comorbidities, the HR was 1.95 . Furthermore, adding the presence of dysphagia to the model confirmed an independent association .Data from 188 patients were available for this analysis. A total of 130 instances of mobility aid uptake were recorded, 81% (52/64) in the anti-cN-1A seropositive group and 63% (78/124) in the seronegative group. The overall median time between disease onset and use of a mobility aid was 8.0 years (IQR 4.6–11.0), with no significant difference between seropositive and seronegative groups (8.0 years (IQR 4.8–10.9), and 6.9 years (IQR 4.4–11.7), respectively; OR 1.01, 95% CI 0.94 to 1.08, p=0.883). Kaplan-Meier curves were not significantly different (log-rank p=0.090), so not shown. In unadjusted analysis, the HR for mobility aid requirement in the antibody-positive group was 1.35 . After adjustment for age at disease onset, gender and comorbidities, the HR for mobility aid requirement was just outside the significance threshold .A significant association between seropositivity for anti-SSB (La) antibodies and anti-cN-1A antibodies was identified  . HoweverWe identified a significant association between an excess of COX-deficient fibres on muscle biopsy and the presence of anti-cN-1A antibodies  . In adjuThis multinational exploratory study represents the first of its kind to combine analysis of clinical, histopathological, other serological and mortality data in a large cohort of patients with IBM stratified according to anti-cN-1A antibody status. Our results will guide future confirmatory studies and highlight potential disease mechanisms warranting further evaluation. We found that the anti-cN-1A antibody positive group had a significantly increased mortality risk independent of age, gender, comorbidities and the presence of dysphagia. We also found a smaller proportion with proximal upper limb weakness at disease onset and an excess of COX-deficient fibres on muscle biopsy in the anti-cN-1A antibody positive group. An increased likelihood of having facial weakness and an association between antibody positivity and death from a respiratory cause was also observed, although the numbers assessed here were small. As in other studies, we did not find a relationship between disease duration and the likelihood of identifying anti-cN-1A antibodies.19et al25There are limited reports in the literature comparing the characteristics of patients with IBM with and without anti-cN-1A antibodies, amounting to 258 patients in four separate studies.The simultaneous discovery of anti-cN-1A antibodies in 2011 by two independent research groups offers potential insights into the pathogenesis of IBM, and will contribute to the debate about the relative influence of the immune system and degeneration.Anti-cN-1A antibodies are present in the sera of 29%–52% of patients with IBM (33% in our cohort).24et alThe higher frequency of COX-negative fibres, a feature of mitochondrial dysfunction, indicates possible differences in molecular pathways within the subgroups defined by anti-cN-1A antibody status. The reasons for increased mortality and the suggestion of increased risk of death from respiratory cause are unexplained, but these findings appear to agree with those of Goyal This study represents the largest cohort of patients with IBM and has only been achieved by an international collaborative effort. Established IBM diagnostic criteria were used to include patients for the analysis, a predefined set of clinical data was retrieved in each patient and all anti-cN-1A testing was performed in one laboratory. However, there remain a number of limitations. The study was retrospective and relied on the identification and recording of clinical characteristics by the treating physicians. In the UK cohort, the recruiting physician (the patient's treating consultant neurologist or rheumatologist) was asked to recall the symptoms that were present at the time of disease onset when completing the pro forma at the time of recruitment, and as such these details may be subject to recall bias. While efforts to minimise missing data were made, data were not complete for all study parameters in all cases, although there was no evidence to suggest that this occurred in a systematic way. Analysis involved pooling of data from different cohorts. There is potential for differences in data collection methodology between cohorts , rather than purely as a diagnostic biomarker. A large prospective study with a sufficient duration of follow-up might offer potential to further investigate the overall utility of anti-cN-1A antibody testing in the clinical and research settings.In this exploratory study, comparison of patients with IBM with and without anti-cN-1A autoantibody reactivity identified differences in their mortality risk, clinical characteristics and histopathological findings. The largest study of its kind has demonstrated that anti-cN-1A antibody testing may, and over and above its diagnostic value, be clinically useful to define distinct IBM subtypes.


Granulocyte colony-stimulating factor (G-CSF)-producing esophageal squamous cell carcinoma (ESCC) has been considered to have a poor prognosis. We successfully treated a case of G-CSF-producing ESCC in a 92-year-old woman.A 92-year-old woman was admitted to our hospital with the complaints of choking while swallowing and dysphagia. Esophagogastroduodenoscopy and contrast-enhanced computed tomography revealed a type 2 esophageal cancer located 26–35 cm from the dental arch, with no distant metastasis. The patient was diagnosed with G-CSF-producing ESCC based on remarkable leukocytosis and high G-CSF levels. The patient underwent radical subtotal esophagectomy. Subsequently, the level of neutrophils  and the level of G-CSF (from 131 to <19.5 pg/mL) decreased significantly. Immunohistochemistry analysis of the resected tissue specimen showed positive staining for G-CSF in the cytoplasm of the tumor cells. Although the patient developed aspiration pneumonitis, after antibiotic treatment, she promptly recovered and was discharged.Herein, we describe a case of successfully treated G-CSF-producing ESCC in a 92-year-old woman. Precise detection and safely performed immediate radical operation are considered essential to achieve a good clinical course. In addition to the mass tumor effects, granulocyte colony-stimulating factor (G-CSF)-producing tumors display additional signs and symptoms of inflammation caused by G-CSF-producing malignant cells . There hWith the aging of the population, the number of oldest old patients with cancer comorbidities has been increasing . TherefoWe report a very rare case of a 92-year-old woman who was promptly diagnosed with G-CSF-producing ESCC and successfully underwent surgical treatment.A 92-year-old woman had a major complaint of choking when swallowing or dysphagia. The patient had been healthy and had no particular medical history besides cataract surgery. She had no history of oral medications, smoking, or alcohol. Another physician had previously attended to her was complaint of choking when swallowing. A narrowing of the lumen of the intrathoracic esophagus was detected by esophagogastroduodenoscopy, and the patient was referred to our hospital for detailed examination.On admission, abnormal symptoms such as fever, anemia, or jaundice were not detected and the performance status was good (score 0 according to the Eastern Cooperative Oncology Group). Laboratory data on admission showed remarkable leukocytosis  and slight decrease in the serum albumin (3.5 g/dL) and C-reactive protein (CRP) levels (1.5 mg/dL). The levels of tumor markers, squamous cell carcinoma antigen (SCC-A), and p53 antibody were high . The respiratory functions and electrocardiograms were within normal ranges. However, the renal function was a slight concern.Esophagogastroduodenoscopy revealed a type 2, circumferential cancer of the esophagus, approximately at 26–35 cm from the dental arch Fig. , and theBased on these findings, the patient was diagnosed with T3N0M0, stage IIA  ESCC. Furthermore, the laboratory data suggested G-CSF-producing carcinoma with serum G-CSF levels of 131 pg/mL.Despite her age, the patient had no comorbidities, and most importantly, she consented to a surgical operation. Therefore, we planned to perform esophagectomy. In Japan, the standard treatment for stage IIA esophageal carcinoma is subtotal esophagectomy with three-field lymph node dissection following preoperative chemotherapy . HoweverIn fact, the subtotal esophagectomy under the right thoracolaparotomy, right lower partial lobectomy, two-field lymph node dissection (instead of three-field), posterior mediastinal route gastric tube reconstruction, and intra-pleural anastomosis were successfully performed. The operation lasted 4 h and 15 min, and the blood loss was 50 mL. The tumor and the right lobe of the lung were attached; therefore, they were resected en bloc because the tumor was considered infiltrative.Histopathological examination of the resected specimen revealed that the primary lesion sized 92 × 54 mm was a moderately differentiated squamous cell carcinoma with two lymph node metastases, and it was diagnosed as a stage III tumor  Fig. . ImmunohAfter the operation, the patient developed aspiration pneumonitis; however, she promptly recovered with the administration of antibiotics. Three weeks after the operation, the leukocyte counts decreased to 5000/μL and the G-CSF levels to <19.5 pg/mL. Thereafter, the patient exhibited a good clinical course and she was discharged on the 29th postoperative day.The patient had neutrophilia without any signs of infection or myeloblast genesis before the operation. After esophagectomy, the number of leukocytes and the level of G-CSF had decreased significantly and the presence of G-CSF was confirmed pathologically. Therefore, the patient was definitively confirmed to have a G-CSF-producing tumor.There have not been any complaints or recurrence, and the patient has remained disease-free from 18 months after the operation until the present day.Robinson first described a G-CSF-producing tumor in 1974 ; howeverG-CSF is a hematopoietic factor produced by the endothelium, macrophages/monocytes, and fibroblasts. It stimulates the bone marrow to produce granulocytes from stem cells and release neutrophils into the bloodstream . It is aThe diagnostic criteria for G-CSF-producing tumors include 1) a marked increase in the leukocyte counts, (2) elevated G-CSF activity, (3) a decrease in leukocyte counts following tumor resection, and (4) the verification of G-CSF production in the tumor [ a markedEsophageal carcinoma is a disease with a poor prognosis . FurtherAccording to Table In addition, with the aging of the population, the chances that we encounter the oldest old patients are increasing . The appIn the present case, the oldest old patient has been alive with a good condition after the operation. In order to improve the quality of life of the oldest old patients, the practical consideration for esophageal carcinoma should be the individualization of therapeutic protocols, tailoring the extent of resection and inclusion or exclusion of preoperative and postoperative procedures. A curative resection with relatively minimal invasion appears to be mandatory for better prognosis with minimal morbidity and mortality in elderly patients.We described a case of successfully treated G-CSF-producing esophageal squamous cell carcinoma in a 92-year-old woman. We assessed the patient’s will and overall condition and chose the best operative method of radical subtotal esophagectomy and could achieve a good clinical course.